Project Summary/Abstract Physiological profiling of Pseudomonas aeruginosa biofilm subpopulations The association of microorganisms into biofilms produces functionally organized microbial structures that promote community survival in a wide range of environments. Much like individual cells within a multicellular organism express different genes from the same DNA base, individual microbial cells located within different regions of a biofilm structure can exhibit distinct genetic programs. These spatially-defined regions of physiologically-differentiated cells are reminiscent of the role of tissues in multicellular organisms, with specific subpopulations in the microbial community serving defined roles to promote the overall health of the biofilm. In this proposal, we seek to assess the function of the subpopulations that we recently identified within the biofilm communities of the pathogenic bacterium Pseudomonas aeruginosa. Based on previous knowledge of the physiological functions of biofilm subpopulations gleaned from studies focused on model microorganisms such as Bacillus subtilis, it is likely that these populations serve one of two major functions: (1) individual resistance to an environmental stressor, enabling subsequent proliferation of single microbial cells within the community to ensure ultimate survival of the microbial strain or (2) the production of shared resources which promote survival of the entire community. The study of subpopulation functionality is particularly important in the context of P. aeruginosa biofilm communities because most infections caused by this organism are mediated by biofilm formation (e.g. infections of wounds, the urinary tract, and the cystic fibrosis lung). The long-term goal of this project is to identify drugs targeting specific biofilm subpopulations. The rationale of this application is that the recent discovery of new P. aeruginosa biofilm subpopulations enables physiological profiling of these populations to identify targetable weaknesses. Our overall hypothesis is that the targeting of subpopulations responsible for producing shared resources can disrupt the entire community to promote clearance of the biofilm. The experiments outlined in this proposal will not only define the community functions of P. aeruginosa subpopulations but will reveal metabolic susceptibilities (the ?Achilles heel?) of these communities, which can be exploited for subsequent therapeutic targeting.